The inhibition of small molecules on pancreatic lipase (PL) has become an important model for screening anti-obesity ingredients in vitro. In this paper， the active constituents with strong binding effect were screened from flavonoids by molecular docking method. The inhibitory activity and type of inhibition were studied by enzyme kinetics， and the binding reaction and mechanism of active constituents and PL were studied by fluorescence spectroscopy. Three flavonoids were selected in vitro: Baicalein (Bai)， Quercetin (Que) and Fisetin (Fis). Molecular docking results showed that the binding of the three flavonoids to PL was located near the residues of Ser153-His264-Asp177 at the catalytic site， and the main forces were hydrophobic interaction and hydrogen bonding. Bai， Que and Fis showed dose-dependent inhibitory activity on PL， with IC50 values of 49.14， 57.78， 61.26 μg/mL， respectively. Inhibition kinetics showed that Bai and Fis had competitive inhibition on PL， while Que was mixed inhibition. Fluorescence spectroscopy showed that the quenching of PL by the three flavonoids belonged to static quenching， and the binding constants were all greater than 105 orders of magnitude， indicating that Bai， Que and Fis formed stable complexes with PL. The analysis of thermodynamic parameters showed that the main binding forces of Bai and PL were hydrogen bond and van der Waals force， and the binding of Que， Fis and PL was through hydrophobic interaction. This paper will provide new ideas for the development of novel PL inhibitors.